Methods of treatment with erythropoietin and albumin fusion protein

ABSTRACT

Biologically active polypeptides comprising a therapeutically active polypeptide fused to human serum albumin or a variant thereof, methods for the preparation thereof, nucleotide sequences encoding such fusion polypeptides, expression cassettes comprising such nucleotide sequences, self-replicating plasmids containing such expression cassettes, and pharmaceutical compositions containing said fusion polypeptides.

This is a divisional of U.S. application Ser. No. 09/984,186, filed Oct.29, 2001 now U.S. Pat. No. 6,686,179, which is a continuation of U.S.application Ser. No. 09/258,532, filed Feb. 26, 1999, now abandoned,which is a divisional of U.S. application Ser. No. 08/797,689, filedJan. 31, 1997, now U.S. Pat. No. 5,876,969, which is a continuation ofU.S. application Ser. No. 08/256,927, filed Jul. 28, 1994, nowabandoned, which is the National Stage Application of InternationalApplication No. PCT/FR93/00085, filed Jan. 28, 1993, which claimsbenefit of French Application 92-01064, filed Jan. 31, 1992, all ofwhich are incorporated herein by reference.

The present invention relates to new biologically active polypeptides,their preparation and pharmaceutical compositions containing them.

More particularly, the present invention relates to essentiallyrecombinant polypeptides composed of an active part derived from anatural or artificial polypeptide having a therapeutic activity andcoupled to an albumin or to a variant of albumin. It is understood thatthe therapeutic activity of the polypeptides of the invention can beeither direct (treatment of diseases), or indirect (and for examplecapable of being used in the prevention of diseases, in the design ofvaccines, in medical imaging techniques and the like).

It is understood in the following text that the albumin variantsdesignate any protein with a high plasma half-life which is obtained bymodification (mutation, deletion and/or addition), by geneticengineering techniques, of a gene encoding a given isomorph of humanserum albumin, as well as any macromolecule with a high plasma half-lifeobtained by in vitro modification of the protein encoded by such genes.Albumin being highly polymorphic, numerous natural variants have beenidentified and classified [Weitkamp L. R. et al., Ann. Hum. Genet. 37(1973) 219].

The aim of the present invention is to prepare artificial proteins whichare biologically active and can be used pharmaceutically. Indeed,numerous polypeptides possessing one or more potential therapeuticactivities cannot be exploited pharmaceutically. This may have variousreasons, such as especially their low stability in vivo, their complexor fragile structure, the difficulty of producing them on anindustrially acceptable scale and the like. Likewise, some polypeptidesdo not give the expected results in vivo because of problems ofadministration, of packaging, of pharmacokinetics and the like.

The present invention makes it possible to overcome these disadvantages.The present invention indeed provides new molecules which permit anoptimal therapeutic exploitation of the biological properties of thesepolypeptides. The present invention results especially from thedemonstration that it is possible to couple genetically any activestructure derived from a biologically active polypeptide to anotherprotein structure consisting of albumin, without impairing the saidbiological properties thereof. It also results from the demonstration bythe Applicant that human serum albumin makes it possible efficiently topresent the active structure to its sites for interaction, and that itprovides a high plasma stability for the polypeptide of the invention.The polypeptides of the invention thus make it possible to maintain, inthe body, a given biological activity for a prolonged period. They thusmake it possible to reduce the administered doses and, in some cases, topotentiate the therapeutic effect, for example by reducing the sideeffects following a higher administration. The polypeptides of theinvention make it possible, in addition, to generate and to usestructures derived from biologically active polypeptides which are verysmall and therefore very specific for a desired effect. It is understoodthat the peptides having a biological activity, which are of therapeuticinterest, may also correspond to non-natural peptide sequences isolatedfor example from random peptide libraries. The polypeptides of theinvention possess, moreover, a particularly advantageous distribution inthe body, which modifies their pharmacokinetic properties and favoursthe development of their biological activity and their use. In addition,they also have the advantage of being weakly or non-immunogenic for theorganism in which they are used. Finally, the polypeptides of theinvention can be expressed (and preferentially secreted) by recombinantorganisms, at levels permitting their industrial exploitation.

One subject of the present invention therefore relates to polypeptidescontaining an active part derived from a polypeptide having atherapeutic activity, coupled to an albumin or a variant of albumin.

In a specific embodiment, the peptides possessing a therapeutic activityare not of human origin. For example, there may be mentioned peptides,or their derivatives, possessing properties which are potentially usefulin the pathologies of the blood and interstitial compartments, such ashirudin, trigramine, antistatine, tick anticoagulant peptides (TAP),arietin, applagin and the like.

More particularly, in the molecules of the invention, the polypeptidehaving a therapeutic activity is a polypeptide of human origin or amolecular variant. For example, this may be all or part of an enzyme, anenzyme inhibitor, an antigen, an antibody, a hormone, a factor involvedin the control of coagulation, an interferon, a cytokine [theinterleukins, but also their variants which are natural antagonists oftheir binding to the receptor(s), the SIS (small induced secreted) typecytokines and for example the macrophage inflammatory proteins (MIPs),and the like], of a growth factor and/or of differentiation [and forexample the transformant growth factors (TGFs), the blood celldifferentiation factors (erythropoietin, M-CSF, G-CSF, GM-CSF and thelike), insulin and the growth factors resembling it (IGFs), oralternatively cell permeability factors (VPF/VEGF), and the like], of afactor involved in the genesis/resorption of bone tissues (OIF andosteospontin for example), of a factor involved in cellular motility ormigration [and for example autocrine motility factor (AMF), migrationstimulating factor (MSF), or alternatively the scatter factor (scatterfactor/hepatocyte growth factor)], of a bactericidal or antifungalfactor, of a chemotactic factor [and for example platelet factor 4(PF4), or alternatively the monocyte chemoattracting peptides (MCP/MCAF)or neutrophil chemoattracting peptides (NCAF), and the like], of acytostatic factor (and for example the proteins which bind togalactosides), of a plasma (and for example von Willebrand factor,fibrinogen and the like) or interstitial (laminin, tenascin, vitronectinand the like) adhesive molecule or extracellular matrices, oralternatively any peptide sequence which is an antagonist or agonist ofmolecular and/or intercellular interactions involved in the pathologiesof the circulatory and interstitial compartments and for example theformation of arterial and venous thrombi, cancerous metastases, tumourangiogenesis, inflammatory shock, autoimmune diseases, bone andosteoarticular pathologies and the like.

The active part of the polypeptides of the invention may consist forexample of the polypeptide having a whole therapeutic activity, or of astructure derived therefrom, or alternatively of a non-naturalpolypeptide isolated from a peptide library. For the purposes of thepresent invention, a derived structure is understood to mean anypolypeptide obtained by modification and preserving a therapeuticactivity. Modification should be understood to mean any mutation,substitution, deletion, addition or modification of genetic and/orchemical nature. Such derivatives may be generated for various reasons,such as especially that of increasing the affinity of the molecule forits binding sites, that of improving its levels of production, that ofincreasing its resistance to proteases, that of increasing itstherapeutic efficacy or alternatively of reducing its side effects, orthat of conferring on it new biological properties. As an example, thechimeric polypeptides of the invention possess pharmacokineticproperties and a biological activity which can be used for theprevention or treatment of diseases.

Particularly advantageous polypeptides of the invention are those inwhich the active part has:

(a) the whole peptide structure or,

(b) a structure derived from (a) by structural modification (mutation,substitution addition and/or deletion of one or more residues) andpossessing a therapeutic activity.

Among the structures of the (b) type, there may be mentioned moreparticularly the molecules in which certain N- or O-glycosylation siteshave been modified or suppressed, the molecules in which one or moreresidues have been substituted, or the molecules in which all thecystein residues have been substituted. There may also be mentionedmolecules obtained from (a) by deletion of regions not involved or nothighly involved in the interaction with the binding sites considered, orexpressing an undesirable activity, and molecules containing, comparedto (a), additional residues such as for example an N-terminal methionineand/or a signal for secretion and/or a joining peptide.

The active part of the molecules of the invention can be coupled eitherdirectly or via an artificial peptide to albumin. Furthermore, it mayconstitute the N-terminal end as well as the C-terminal end of themolecule. Preferably, in the molecules of the invention, the active partconstitutes the C-terminal part of the chimera. It is also understoodthat the biologically active part may be repetitive within the chimera.A schematic representation of the molecules of the invention is given inFIG. 1.

Another subject of the invention relates to a process for preparing thechimeric molecules described above. More specifically, this processconsists in causing a eukaryotic or prokaryotic cellular host to expressa nucleotide sequence encoding the desired polypeptide, and then inharvesting the polypeptide produced.

Among the eukaryotic hosts which can be used within the framework of thepresent invention, there may be mentioned animal cells, yeasts or fungi.In particular, as regards yeasts, there may be mentioned yeasts of thegenus Saccharomyces, Kluyveromyces, Pichia, Schwanniomyces, orHansenula. As regards animal cells, there may be mentioned COS, CHO andC127 cells and the like. Among the fungi capable of being used in thepresent invention, there may be mentioned more particularly Aspergillusssp, or Trichoderma ssp. As prokaryotic hosts, the use of bacteria suchas Escherichia coli,or belonging to the genera Corynebacterium,Bacillus, or Streptomyces is preferred.

The nucleotide sequences which can be used within the framework of thepresent invention can be prepared in various ways. Generally, they areobtained by assembling, in reading phase, the sequences encoding each ofthe functional parts of the polypeptide. The latter may be isolated bythe techniques of persons skilled in the art, and for example directlyfrom cellular messenger RNAs (mRNAs), or by recloning from acomplementary DNA (cDNA) library, or alternatively they may becompletely synthetic nucleotide sequences. It is understood,furthermore, that the nucleotide sequences may also be subsequentlymodified, for example by the techniques of genetic engineering, in orderto obtain derivatives or variants of the said sequences.

More preferably, in the process of the invention, the nucleotidesequence is part of an expression cassette comprising a region forinitiation of transcription (promoter region) permitting, in the hostcells, the expression of the nucleotide sequence placed under itscontrol and encoding the polypeptides of the invention. This region maycome from promoter regions of genes which are highly expressed in thehost cell used, the expression being constitutive or regulatable. Asregards yeasts, it may be the promoter of the gene for phosphoglyceratekinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GPD), lactase(LAC4), enolases (ENO), alcohol dehydrogenases (ADH), and the like. Asregards bacteria, it may be the promoter of the right-hand or left-handgenes from the lambda bacteriophage (PL, PR), or alternatively thepromoters of the genes for the tryptophan (Ptrp) or lactose (Plac)operons. In addition, this control region can be modified, for exampleby in vitro mutagenesis, by the introduction of additional controlelements or of synthetic sequences, or by deletions or substitutions ofthe original control elements. The expression cassette may also comprisea region for termination of transcription which is functional in thehost envisaged, positioned immediately downstream of the nucleotidesequence encoding a polypeptide of the invention.

In a preferred mode, the polypeptides of the invention result from theexpression, in a eukaryotic or prokaryotic host, of a nucleotidesequence and from the secretion of the product of expression of the saidsequence into the culture medium. It is indeed particularly advantageousto be able to obtain, by the recombinant route, molecules directly inthe culture medium. In this case, the nucleotide sequence encoding apolypeptide of the invention is preceded by a “leader” sequence (orsignal sequence) directing the nascent polypeptide in the secretorypathways of the host used. This “leader” sequence may be the naturalsignal sequence of the biologically active polypeptide in the case wherethe latter is a naturally secreted protein, or that of the stabilizingstructure, but it may also be any other functional “leader” sequence, oran artificial “leader” sequence. The choice of one or the other of thesesequences is especially guided by the host used. Examples of functionalsignal sequences include those of the genes for the sexual pheromones orthe “killer” toxins of yeasts.

In addition to the expression cassette, one or several markers whichmake it possible to select the recombinant host may be added, such asfor example the URA3 gene from the yeast S. cerevisiae,or genesconferring the resistance to antibiotics such as genetic in (G418) or toany other toxic compound such as certain metal ions.

The unit formed by the expression cassette and by the selectable markercan be introduced directly into the considered host cells, or previouslyinserted in a functional self-replicating vector. In the first case,sequences homologous to regions present in the genome of the host cellsare preferably added to this unit; the said sequences then beingpositioned on each side of the expression cassette and of the selectablegene so as to increase the frequency of integration of the unit into thegenome of the host by targetting the integration of the sequences byhomologous recombination. In the case where the expression cassette isinserted in a replicative system, a preferred replication system foryeasts of the genus Kluyveromyces is derived from the plasmid pKD1originally isolated from K. drosophilarum; a preferred replicationsystem for yeasts of the genus Saccharomyces is derived from the 2μplasmid from S. cerevisiae. Furthermore, this expression plasmid maycontain all or part of the said replication systems, or may combineelements derived both from the plasmid pKD1 and the 2μ plasmid.

In addition, the expression plasmids may be shuttle vectors between abacterial host such as Escherichia coli and the chosen host cell. Inthis case, a replication origin and a selectable marker functioning inthe bacterial host are required. It is also possible to positionrestriction sites surrounding the bacterial and unique sequences on theexpression vector: this makes it possible to suppress these sequences bycutting and religation in vitro of the truncated vector beforetransformation of the host cells, which may result in an increase in thenumber of copies and in an increased stability of the expressionplasmids in the said hosts. For example, such restriction sites maycorrespond to sequences such as 5′-GGCCNNNNNGGCC-3′ SEQ ID NO:19 (SfiI)or 5′-GCGGCCGC-3′ (NotI) in so far as these sites are extremely rare andgenerally absent from an expression vector.

After construction of such vectors or expression cassette, the latterare introduced into the host cells selected according to theconventional techniques described in the literature. In this respect,any method permitting the introduction of a foreign DNA into a cell canbe used. This may be especially transformation, electroporation,conjugation, or any other technique known to persons skilled in the art.As an example of yeast-type hosts, the various strains of Kluyveromycesused were transformed by treating the whole cells in the presence oflithium acetate and polyethylene glycol, according to the techniquedescribed by Ito et al. [J. Bacteriol. 153 (1983) 163]. Thetransformation technique described by Durrens et al. [Curr. Genet. 18(1990) 7] using ethylene glycol and dimethyl sulphoxide was also used.It is also possible to transform the yeasts by electroporation,according to the method described by Karube et al. [FEBS Letters 182(1985) 90]. An alternative procedure is also described in detail in theexamples below.

After selection of the transformed cells, the cells expressing the saidpolypeptides are inoculated and the recovery of the said polypeptidescan be carried out, either during the cell growth for the “continuous”processes, or at the end of growth for the “batch” cultures. Thepolypeptides which are the subject of the present invention are thenpurified from the culture supernatant for their molecular,pharmacokinetic and biological characterization.

A preferred expression system for the polypeptides of the inventionconsists in using yeasts of the genus Kluyveromyces as host cell,transformed by certain vectors derived from the extrachromosomalreplicon pKD1 originally isolated from K. marxianus var. drosophilarum.These yeasts, and in particular K. lactis and K. fragilis are generallycapable of stably replicating the said vectors and possess, in addition,the advantage of being included in the list of G.R.A.S. (“GenerallyRecognized As Safe”) organisms. Favoured yeasts are preferablyindustrial yeasts of the genus Kluyveromyces which are capable of stablyreplicating the said plasmids derived from the plasmid pKD1 and in whichhas been inserted a selectable marker as well as an expression cassettepermitting the secretion, at high levels, of the polypeptides of theinvention.

The present invention also relates to the nucleotide sequences encodingthe chimeric polypeptides described above, as well as the eukaryotic orprokaryotic recombinant cells comprising such sequences.

The present invention also relates to the application, as medicinalproducts, of the polypeptides according to the present invention. Moreparticularly, the subject of the invention is any pharmaceuticalcomposition comprising one or more polypeptides or nucleotide sequencesas described above. The nucleotide sequences can indeed be used in genetherapy.

The present invention will be more fully described with the aid of thefollowing examples, which should be considered as illustrative andnon-limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The representations of the plasmids indicated in the following figuresare not plotted to scale and only the restriction sites important forthe understanding of the clonings carried out have been indicated.

FIG. 1A is a schematic representation of the chimera of the HSA-PEPTIDEtype;

FIG. 1B is a schematic representation of a chimera of the PEPTIDE-HSAtype; and

FIG. 1C is a schematic representation of a chimera of thePEPTIDE-HSA-PEPTIDE type. Abbreviations used: M/LP, translationalinitiator methionine residue, optionally followed by a signal sequencefor secretion; HSA, mature albumin or one of its molecular variants;PEP, peptide of natural or artificial origin possessing a giventherapeutic property. The PEP sequence may be present several times inthe FIG. 1A, B or C molecules. The black arrow indicates the N-terminalend of the mature protein.

FIGS. 2(a) and 2(c), together, comprise an example of a nucleotidesequence (SEQ ID NO:1) and an amino acid sequence (SEQ ID NO:2) of aHindIII restriction fragment encoding a chimeric protein of theprepro-HSA-PEPTIDE type. The black arrows indicate the end of the “pre”and “pro” regions of HSA. The MstII restriction site is underligned andthe codon specifying the termination of translation is in boldcharacters.

FIG. 3: Restriction map for the plasmid pYG105 and generic strategy forconstruction of the plasmids for expression of the chimeric proteins ofthe present invention. Abbreviations used: P, transcriptional promoter;T, transcriptional terminator; IR, inverted repeat sequences of theplasmid pKD1; LP, signal sequence for secretion; Apr and Kmr designatethe genes for resistance to ampicillin (E. coli) and to G418 (yeasts),respectively.

FIGS. 4A, 4B, 4C, 4D, 4E and 4F collectively show examples of nucleotidesequences of MstII-HindIII restriction fragments derived from the vonWillebrand factor. FIG. 4A is a representation of the structure of theMstII-HindIII fragment of the plasmid pYG1248 (SEQ ID NOS:3 and 4). FIG.4B is a representation of the structure of the MstII-HindIII fragment ofthe plasmid pYG1214 (SEQ ID NOS:5 and 6). FIG. 4C is a representation ofthe MstII-HindIII fragment of the plasmid pYG1206; in this particularchimera, the Leu694 residue of the vWF is also the last residue (Leu585)of the HSA. FIG. 4D is a representation of the MstII-HindIII fragment ofthe plasmid pYG1223 (SEQ ID NOS:9 and 10). The numbering of the aminoacids corresponds to the numbering of the mature vWF according to Titaniet al. [Biochemistry 25 (1986) 3171-3184]. The MstII and HindIIIrestriction sites are underlined and the translation termination codonis in bold characters. FIGS. 4E and 4F show a nucleotide sequence (SEQID NO:3) of the MstII-HindIII restriction fragment of the plasmidpYG1248. The numbering of the amino acids (right-hand column)corresponds to the mature chimeric protein HSA-vWF470→713 (829residues). The Thr470, Leu494, Asp498, Pro502, Tyr508, Leu694, Pro704and Pro708 residues of the mature vWF are underlined.

FIGS. 5A, 5B, and 5C collectively show the characterization of thematerial secreted after 4 days of culture (erlenmeyers) of the strainCBS 293.91 transformed with the plasmids pYG1248 (plasmid for expressionof a chimera of the HSA-vWF Thr470→Val713) and pKan707 (controlplasmid). In this experiment, the polypeptides for FIGS. 5A, 5B, and 5Cwere run on the same gel (8.5% SDS-PAGE) and then treated separately.

FIG. 5A shows the results of coomassie blue staining of a molecularweight standard (lane 2); of a supernatant equivalent to 50 μl of theculture transformed with the plasmid pKan707 in YPL medium (lane 1); theplasmid pYG1248 in YPD medium (lane 3) and the plasmid pYG1248 in YPLmedium (lane 4).

FIG. 5B shows the results of immunological characterization of thesecreted material after using mouse antibodies directed against humanvWF. The lanes are the same as described for FIG. 5A except thatbiotinilated molecular weight standards were used (lane 2).

FIG. 5C shows the results of immunological characterization of thesecreted material after using rabbit antibodies directed against humanalbumin: supernatant equivalent to 50 μl of the culture transformed withthe plasmid pKan707 in YPL medium (lane 1), the plasmid pYG1248 in YPDmedium (lane 2) the plasmid pYG1248 in YPL medium (lane 3).

FIGS. 6A and 6B show the kinetic analysis of secretion of a chimera ofthe invention by the strain CBS 293.91 transformed with the plasmidpYG1206 (HSA-vWF Leu694-Pro708).

In FIG. 6A, coomassie blue staining was employed. Lane 1 is themolecular weight standard, lane 2 is the supernatant equivalent to 2.5μl of a “Fed Batch” culture in YPD medium after 24 hours of growth; lane3 is the supernatant of the same culture after 40 hours; and lane 4 isthe supernatant of the same culture after 46 hours of growth.

FIG. 6B shows the results of immunological characterization of thesecreted material after using mouse antibodies directed against thehuman vWF. The lanes are the same as in FIG. 6A except that biotinilatedmolecular weight standards were used.

FIG. 7: Characterization of the material secreted by K. lactistransformed with the plasmids pKan707 (control plasmid, lane 2), pYG1206(lane 3), pYG1214 (lane 4) and pYG1223 (lane 5); molecular weightstandard (lane 1). The deposits correspond to 50 μl of supernatant froma stationary culture after growing in YPD medium, running on an 8.5%acrylamide gel and staining with coomassie blue.

FIG. 8: Nucleotide sequence (SEQ ID NO:11) and amino acid sequence (SEQID NO:12) of the MstII-HindIII restriction fragment of the plasmidpYG1341 (HSA-UK1→135). The limit of the EGF-like domain (UK1→46) presentin the MstII-HindIII restriction fragment of the plasmid pYG1340 isindicated. The numbering of the amino acids corresponds to the maturechimeric protein SAU-UK1→135 (720 residues).

FIG. 9: Secretion of the HSA-UK1-46 and HSA-UK1-135 chimeras by thestrain CBS 293.91 respectively transformed with the plasmids pYG1343(HSA-UK1-46) and pYG1345 (HSA-UK1-135), after 4 days of growth (YPL+G418medium). The deposits (equivalent to 50 μl of culture) are run on an8.5% PAGE-SDS gel and stained with coomassie blue: supernatant from aclone transformed with the plasmids pKan707 (lane 1), pYG1343 (lane 3)or pYG1345 (lane 4); molecular weight standard (lane 2).

FIG. 10: Nucleotide sequence (SEQ ID NO:13) and amino acid sequence (SEQID NO:14) of the MstII-HindIII restriction fragment of the plasmidpYG1259 (HSA-G.CSF). The limit of the G-CSF part (174 residues) isindicated. The ApaI and SstI (SstI) restriction sites are underlined.The numbering of the amino acids corresponds to the mature chimericprotein HSA-G.CSF (759 residues).

FIGS. 11(a)-(d) together comprise the nucleotide sequence (SEQ ID NO:15)and amino acid sequence (SEQ ID NO:16) of the HindIII restrictionfragment of the plasmid pYG1301 (chimera G.CSF-Gly4-HSA). The blackarrows indicate the end of the “pre” and “pro” regions of HSA. The ApaI,SstI (SacI) and MstII restriction sites are underlined. The G.CSF (174residues) and HSA (585 residues) domains are separated by the syntheticlinker GGGG. The numbering of the amino acids corresponds to the maturechimeric protein G.CSF-Gly4-SAH (763 residues). The nucleotide sequencebetween the translation termination codon and the HindIII site comesfrom the HSA complementary DNA (cDNA) as described in Patent ApplicationEP 361 991.

FIGS. 12A, 12B, and 12C collectively show the characterization of thematerial secreted after 4 days of culture (erlenmeyers) of the strainCBS 293.91 transformed with the plasmids pYG1266 (plasmid for expressionof a chimera of the HSA-G.CSF type) and pKan707 (control plasmid). Inthis experiment, the polypeptides for FIGS. 12A, 12B, 12C were run onthe same gel (8.5% SDS-PAGE) and then treated separately.

FIG. 12A shows the results of coomassie blue staining of a molecularweight standard (lane 2); supernatant equivalent to 100 μl of culturetransformed with the plasmid pKan707 in YPL medium (lane 1); the plasmidpYG1266 in YPD medium (lane 3) and the plasmid pYG1266 in YPL medium(lane 4).

FIG. 12B shows the results of immunological characterization of thematerial secreted after using primary antibodies directed against humanG-CSF. The lanes are as described above for FIG. 12A.

FIG. 12C shows the results of immunological characterization of thematerial secreted after using primary antibodies directed against humanalbumin. The lanes are as described above for FIG. 12A.

FIGS. 13A and B collectively show the characterization of the materialsecreted after 4 days of culture (erlenmeyers in YPD medium) of thestrain CBS 293.91 transformed with the plasmids pYG1267 (chimeraHSA-G.CSF), pYG1303 (chimera G.CSF-Gly4-HSA) and pYG1352 (chimeraHSA-Gly4-G.CSF) after running on an 8.5% SDS-PAGE gel.

FIG. 13A shows the results of coomassie blue staining of a supernatantequivalent to 100 μl of the culture transformed with the plasmid pYG1303(lane 1), the plasmid pYG1267 (lane 2), and the plasmid pYG1352 (lane3). Lane 4 is the molecular weight standard.

B, immunological characterization of the material secreted after usingprimary antibodies directed against the human G-CSF: same legend as inA.

FIG. 14: Nucleotide sequence (SEQ ID NO:17) and amino acid sequence (SEQID NO:18) of the MstII-HindIII restriction fragment of the plasmidpYG1382 (HSA-Fv′). The VH (124 residues) and VL (107 residues) domainsof the Fv′ fragment are separated by the synthetic linker (GGGGS)×3. Thenumbering of the amino acids corresponds to the mature chimeric proteinHSA-Fv′ (831 residues).

FIGS. 15A and 15B collectively show the characterization of thesecretions of the chimera HSA-Fv′ by the strain CBS 293.91 transformedwith the plasmid pYG1383 (LAC4) after 4 days of growth in erlenmeyers at28° C. in YPD medium (lane 2), and in YPL medium (lane 3). Lane 1 showsthe molecular weight standard. The deposits, equivalent to 200 μl ofculture (precipitation with ethanol), are run on a PAGE-SDS gel (8.5%).

FIG. 15A shows the results of coomassie blue staining of the gel.

FIG. 15B shows the results of immunological characterization of thematerial secreted after using primary antibodies directed against HSA.

FIG. 16: Assay of the in vitro antagonistic activity of theagglutination of human platelets fixed with formaldehyde: IC50 of thehybrids HSA-vWF694-708, [HSA-vWF470-713 C471G, C474G] and[HSA-vWF470-704 C471G, C474G] compared with the standard RG12986. Thedetermination of the dose-dependent inhibition of the plateletagglutination is carried out according to the method described by C.Prior et al. [Bio/Technology (1992) 10 66] using an aggregameterrecording the variations in optical transmission, with stirring, at 37°C. in the presence of human vWF, botrocetin (8.2 mg/ml) of the testproduct at various dilutions. The concentration of the product whichmakes it possible to inhibit the control agglutination (in the absenceof product) by half is then determined (IC50).

FIG. 17: Activity on the in vitro cellular proliferation of the murineline NFS60. The radioactivity (3 H-thymidine) incorporated into thecellular nuclei after 6 hours of incubation is represented on the y-axis(cpm); the quantity of product indicated on the x-axis is expressed inmolarity (arbitrary units).

FIG. 18: Activity on granulopoiesis in vivo in rats. The number ofneutrophils (average for 7 animals) is indicated on the y-axis as afunction of time. The products tested are the chimera HSA-G.CSF(pYG1266), 4 or 40 mg/rat/day), the reference G-CSF (10 mg/rat/day), therecombinant HSA purified from Kluyveromyces lactis supernatant (HSA, 30mg/rat/day, cf. EP 361 991), or physiological saline.

EXAMPLES General Cloning Techniques

The methods conventionally used in molecular biology, such as thepreparative extractions of plasmid DNA, the centrifugation of plasmidDNA in caesium chloride gradient, electrophoresis on agarose oracrylamide gels, purification of DNA fragments by electroelution,extractions of proteins with phenol or phenol-chloroform, DNAprecipitation in saline medium with ethanol or isopropanol,transformation in Escherichia coli,and the like are well known topersons skilled in the art and are widely described in the literature[Maniatis T. et al., “Molecular Cloning, a Laboratory Manual”, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982; Ausubel F. M.et al. (eds), “Current Protocols in Molecular Biology”, John Wiley &Sons, New York, 1987].

The restriction enzymes were provided by New England Biolabs (Biolabs),Bethesda Research Laboratories (BRL) or Amersham and are used accordingto the recommendations of the suppliers.

The pBR322 and pUC type plasmids and the phages of the M13 series are ofcommercial origin (Bethesda Research Laboratories).

For the ligations, the DNA fragments are separated according to theirsize by electrophoresis on agarose or acrylamide gels, extracted withphenol or with a phenol/chloroform mixture, precipitated with ethanoland then incubated in the presence of phage T4 DNA ligase (Biolabs)according to the recommendations of the manufacturer.

The filling of the protruding 5′ ends is carried out by the Klenowfragment of DNA polymerase I of E. coli (Biolabs) according to thespecifications of the supplier. The destruction of the protruding 3′ends is carried out in the presence of phage T4 DNA polymerase (Biolabs)used according to the recommendations of the manufacturer. Thedestruction of the protruding 5′ ends is carried out by a controlledtreatment with S1 nuclease.

Site-directed mutagenesis in vitro with synthetic oligodeoxynucleotidesis carried out according to the method developed by Taylor et al.[Nucleic Acids Res. 13 (1985) 8749-8764] using the kit distributed byAmersham.

The enzymatic amplification of DNA fragments by the so-called PCRtechnique [Polymerase-catalyzed Chain Reaction, Saiki R. K. et al.,Science 230 (1985) 1350-1354; Mullis K. B. and Faloona F. A., Meth.Enzym. 155 (1987) 335-350] is carried out using a “DNA thermal cycler”(Perkin Elmer Cetus) according to the specifications of themanufacturer.

The verification of the nucleotide sequences is carried out by themethod developed by Sanger et al. [Proc. Natl. Acad. Sci. U.S.A., 74(1977) 5463-5467] using the kit distributed by Amersham.

The transformations of K. lactis with DNA from the plasmids forexpression of the proteins of the present invention are carried out byany technique known to persons skilled in the art, and of which anexample is given in the text.

Except where otherwise stated, the bacterial strains used are E. coli MC1060 (lacIPOZYA, X74,galU, galK, strAr), or E. coli TG1 (lac, proA,B,supE, thi, hsdD5/FtraD36, proA+ B+, lacIq, lacZ, M15).

The yeast strains used belong to the budding yeasts and moreparticularly to yeasts of the genus Kluyveromyces. The K. lactis MW98-8C(a, uraA, arg, lys, K+, pKD1°) and K. lactis CBS 293.91 strain wereparticularly used; a sample of the MW98-8C strain was deposited on 16Sep. 1988 at Centraalbureau voor Schimmelkulturen (CBS) at Baam (theNetherlands) where it was registered under the number CBS 579.88.

A bacterial strain (E. coli) transformed with the plasmid pET-8c52K wasdeposited on 17 Apr. 1990 with the American Type Culture Collectionunder the number ATCC 68306.

The yeast strains transformed with the expression plasmids encoding theproteins of the present invention are cultured in erlenmeyers or in 21pilot fermenters (SETRIC, France) at 28° C. in rich medium (YPD: 1%yeast extract, 2% Bactopeptone, 2% glucose; or YPL: 1% yeast extract, 2%Bactopeptone, 2% lactose) with constant stirring.

Example 1 Coupling at the C-Terminus of HSA

The plasmid pYG404 is described in Patent Application EP 361 991. Thisplasmid contains a HindIII restriction fragment encoding the prepro-HSAgene preceded by the 21 nucleotides naturally present immediatelyupstream of the initiator ATG for translation of the PGK gene of S.cerevisiae. The nucleotide sequence of this restriction fragment isincluded in that of FIG. 2. The MstII site localized in the codingsequence, three residues from the codon specifying the end oftranslation is particularly useful as site for cloning a biologicallyactive peptide which it is desired to couple in translational phase atthe C-terminus of HSA. In a specific embodiment, it is useful to usepeptides whose sequence is encoded by an MstII-HindIII restrictionfragment of the type: 5′-CCTTAGGCTTA [3×N]p TAAGCTT-3′ (SEQ ID NO:20),the sequence encoding the biologically active peptide (p residues) is[3×N]p). The ligation of this fragment to the HindIII-MstII restrictionfragment corresponding to the entire gene encoding HSA, with theexception of the three C-terminalmost amino acids (leucin-glycine-leucinresidues) generates a HindIII restriction fragment containing a hybridgene encoding a chimeric protein of the HSA-PEPTIDE type (FIG. 1, panelA), immediately preceded by the “prepro” export region of HSA. Inanother embodiment, the biologically active peptide may be present morethan once in the chimera.

Example 2 Coupling at the N-Terminus of HSA

In a specific embodiment, the combined techniques of site-directedmutagenesis and PCR amplification make it possible to construct hybridgenes encoding a chimeric protein resulting from the translationalcoupling between a signal peptide (and for example the prepro region ofHSA), a sequence including the biologically active peptide and themature form of HSA or one of its molecular variants. These hybrid genesare preferably bordered in 5′ of the translational initiator ATG and in3′ of the translational stop codon by HindIII restriction sites andencode chimeric proteins of the PEPTIDE-HSA type (FIG. 1, panel B). In astill more specific embodiment, the biologically active peptide may bepresent more than once in the chimera.

Example 3 Coupling at the N- and C-Terminus of HSA

The combined techniques of site-directed mutagenesis and PCRamplification described in Examples 1 and 2 make it possible toconstruct hybrid genes encoding a chimeric protein resulting from thetranslational coupling between the mature form of HSA, or one of itsmolecular variants, and a biologically active peptide coupled to the N-and C-terminal ends of HSA. These hybrid genes are preferably borderedin 5′ of the translational initiator ATG and in 3′ of the translationalstop codon by HindIII restriction sites and encode chimeric proteins ofthe PEPTIDE-HSA-PEPTIDE type (FIG. 1, panel C), immediately preceded bythe “prepro” export region of HSA. In a still more specific embodiment,the biologically active peptide may be present more than once in thechimera.

Example 4 Expression Plasmids

The chimeric proteins of the preceding examples can be expressed inyeasts using functional, regulatable or constitutive promoters such as,for example, those present in the plasmids pYG105 (LAC4 promoter ofKluyveromyces lactis), pYG106 (PGK promoter of Saccharomycescerevisiae), pYG536 (PHO5 promoter of S. cerevisiae), or hybridpromoters such as those described in Patent Application EP 361 991. Theplasmids pYG105 and pYG106 are particularly useful here because theypermit the expression of the genes encoded by the HindIII restrictionfragments as described in the preceding examples and cloned into theHindIII site and in the productive orientation (defined as theorientation which places the “prepro” region of albumin proximallyrelative to the promoter for transcription), using promoters which arefunctional in K. lactis, regulatable (pYG105) or constitutive (pYG106).The plasmid pYG105 corresponds to the plasmid pKan707 described inPatent Application EP 361 991 in which the HindIII restriction sitewhich is unique and localized in the gene for resistance to geneticin(G418) has been destroyed by site-directed mutagenesis while preservingan unchanged protein (oligodeoxynucleotide5′-GAAATGCATAAGCTCTTGCCATTCTCACCG-3′)(SEQ ID NO:21). The SalI-SacIfragment encoding the URA3 gene of the mutated plasmid was then replacedwith a SalI-SacI restriction fragment containing an expression cassetteconsisting of the LAC4 promoter of K. lactis (in the form of aSalI-HindIII fragment) and the terminator of the PGK gene of S.cerevisiae (in the form of a HindIII-SacI fragment). The plasmid pYG105is mitotically very stable in the Kluyveromyces yeasts and a restrictionmap thereof is given in FIG. 3. The plasmids pYG105 and pYG106 differfrom each other only in the nature of the promoter for transcriptionencoded by the SalI-HindIII fragment.

Example 5 Transformation of the Yeasts

The transformation of the yeasts belonging to the genus Kluyveromyces,and in particular the strains MW98-8C and CBS 293.91 of K. lactis iscarried out for example by the technique for treating whole cells withlithium acetate [Ito H. et al., J. Bacteriol. 153 (1983) 163-168],adapted as follows. The growth of the cells is carried out at 28° C. in50 ml of YPD medium, with stirring and up to an optical density of 600nm (OD600) of between 0.6 and 0.8; the cells are harvested bycentrifugation at low speed, washed in a sterile solution of TE (10 MMTris HCl pH 7.4; 1 mM EDTA), resuspended in 3-4 ml of lithium acetate(0.1M in TE) in order to obtain a cellular density of about 2×10⁸cells/ml, and then incubated at 30° C. for 1 hour with moderatestirring. Aliquots of 0.1 ml of the resulting suspension of competentcells are incubated at 30° C. for 1 hour in the presence of DNA and at afinal concentration of 35% polyethylene glycol (PEG4000, Sigma). After aheat shock of 5 minutes at 42° C., the cells are washed twice,resuspended in 0.2 ml of sterile water and incubated for 16 hours at 28°C. in 2 ml of YPD medium in order to permit the phenotypic expression ofthe gene for resistance to G418 expressed under the control of the Pk1promoter (cf. EP 361 991); 200 μl of the cellular suspension are thenplated on selective YPD dishes (G418, 200 μg/ml). The dishes areincubated at 28° C. and the transformants appear after 2 to 3 days ofcell growth.

Example 6 Secretion of the Chimeras

After selection on rich medium supplemented with G418,the recombinantclones are tested for their capacity to secrete the mature form of thechimeric proteins. Few clones, corresponding to the strain CBS 293.91 orMW98-8C transformed by the plasmids for expression of the chimerasbetween HSA and the biologically active part, are incubated in YPD orYPL medium at 28° C. The cellular supernatants are recovered bycentrifugation when the cells reach the stationary growth phase,optionally concentrated 10 times by precipitation for 30 minutes at −20°C. in a final concentration of 60% ethanol, and then tested afterelectrophoresis on an 8.5% SDS-PAGE gel, either directly by staining thegel with coomassie blue, or after immunoblotting using primaryantibodies directed against the biologically active part or a rabbitpolyclonal serum directed against HSA. During the experiments forimmunological detection, the nitrocellulose filter is first incubated inthe presence of specific primary antibodies, washed several times,incubated in the presence of goat antibodies directed against theprimary antibodies, and then incubated in the presence of anavidin-peroxidase complex using the “ABC kit” distributed by Vectastain(Biosys S. A., Compiegne, France). The immunological reaction is thenrevealed by the addition of 3,3′-diamino benzidine tetrahydrochloride(Prolabo) in the presence of hydrogen peroxide, according to therecommendations of the manufacturer.

Example 7 Chimeras Derived from the Von Willebrand Factor

E.7.1. Fragments Antagonizing the Binding of vWF to the Platelets

E. 7.1.1. Thr470-Val713 Residues of vWF

The plasmid pET-8c52K contains a fragment of the vWF cDNA encodingresidues 445 to 733 of human vWF and therefore includes several crucialdeterminants of the interaction between vWF and the platelets on the onehand, and certain elements of the basal membrane and the sub-endothelialtissue on the other, and especially the peptides G10 and D5 whichantagonize the interaction between vWF and GP1b [Mori H. et al., J.Biol. Chem. 263 (1988) 17901-17904]. This peptide sequence is identicalto the corresponding sequence described by Titani et al. [Biochemistry25,(1986) 3171-3184]. The amplification of these genetic determinantscan be carried out using the plasmid pET-8c52K, for example by the PCRamplification technique, using as primer oligodeoxynucleotides encodingcontiguous residues localized on either side of the sequence to beamplified. The amplified fragments are then cloned into vectors of theM13 type for their verification by sequencing using either the universalprimers situated on either side of the multiple cloning site, oroligodeoxynucleotides specific for the amplified region of the vWF geneof which the sequence of several isomorphs is known [Sadler J. E. etal., Proc. Natl. Acad. Sci. 82 (1985) 6394-6398; Verweij C. L. et al.,EMBO J. 5 (1986) 1839-1847; Shelton-Inloes B. B. et al., Biochemistry 25(1986) 3164-3171; Bonthron D. et al., Nucleic Acids Res. 17 (1986)7125-7127]. Thus, the PCR amplification of the plasmid pET-8c52K withthe oligodeoxynucleotides 5′-CCCGGGATCCCTTAGGCTTAACCTGTGAAGCCTGC-3′ (SEQID NO:22) (Sq1969,the MstII site is underlined) and 5′-CCCGGGATCCAAGCTTAGACTTGTGCCATGTCG-3′ (SEQ ID NO:23) (Sq2029,the HindIII site isunderlined) generates an MstII-HindIII restriction fragment includingthe Thr470 to Val713 residues of vWF (FIG. 4, panel E). The ligation ofthis fragment to the HindIII-MstII restriction fragment corresponding tothe entire gene encoding HSA, with the exception of the threeC-terminalmost amino acids (cf. FIG. 2) generates a HindIII restrictionfragment containing a hybrid gene encoding a chimeric protein of theHSA-PEPTIDE type (FIG. 1, panel A), immediately preceded by the “prepro”export region of HSA. This restriction fragment is cloned in theproductive orientation and into the HindIII site of the plasmidpYG105,which generates the expression plasmid pYG1248 (HSA-vWF470-713).

E.7.1.2. Molecular Variants:

In another embodiment, the binding site of vWF is a peptide includingthe Thr470 to Asp498 residues of the mature vWF. This sequence includingthe peptide G10 (Cys474-Pro488) described by Mori et al. [J. Biol. Chem.263 (1988) 17901-17904] and capable of antagonizing the interaction ofhuman vWF with the GP1b of the human platelets. The sequencecorresponding to the peptide G10 is first included in an MstII-HindIIIrestriction fragment (FIG. 4, panel B), for example by PCR amplificationof the plasmid pET-8c52K with the oligodeoxynucleotides Sq1969 and5′-CCCGGGATCCAAGCTTAGTCCTCCACATACAG-3′ (SEQ ID NO:24) (Sq1970,theHindIII site is underlined), which generates an MstII-HindIIIrestriction fragment including the peptide G10,and whose sequence is:5′-CCTTAGGCTTAACCTGTGAAGCCTGCCAGGAGCCGGGAGGCCTGGTGGTGCCTCCCACAGATGCCCCGGTGAGCCCC-ACCACTCTGTATGTGGAGGACTAAGCTT-3′ (SEQ ID NO:25) (thesequence encoding the peptide G10 is in bold characters). The ligationof this fragment to the HindIII-MstII restriction fragment correspondingto the entire gene encoding HSA, with the exception of the threeC-terminalmost amino acids (cf. FIG. 2) generates a HindIII restrictionfragment containing a hybrid gene encoding a chimeric protein of theHSA-PEPTIDE type (FIG. 1, panel A), immediately preceded by the “prepro”export region of HSA. This restriction fragment is cloned in theproductive orientation into the HindIII site of the plasmid pYG105,whichgenerates the expression plasmid pYG1214.

In another embodiment, the site for binding of vWF to GP1b is directlydesigned with the aid of synthetic oligodeoxynucleotides, and forexample the oligodeoxynucleotides5′-TTAGGCCTCTGTGACCTTGCCCCTGAAGCCCCTCCTCCTACTCTGCCCCCCTAAGCTT A-3′ (SEQID NO:26) and 5′-GATCTAAGCTTAGGGGGGCAGAGTAGGAGGAGGGGCTTCAGGGGCAAGGTCACAGAGGCC-3′ (SEQ ID NO:27). These oligodeoxynucleotides form, by pairing, aMstII-BgIII restriction fragment including the MstII-HindIII fragment(FIG. 4, panel C) corresponding to the peptide D5 defined by the Leu694to Pro708 residues of vWF. The ligation of the MstII-HindIII fragment tothe HindIII-MstII restriction fragment corresponding to the entire geneencoding HSA with the exception of the three C-terminalmost amino acids(cf. FIG. 2) generates a HindIII restriction fragment containing ahybrid gene encoding a chimeric protein of the HSA-PEPTIDE type (FIG. 1,panel A), immediately preceded by the “prepro” export region of HSA.This restriction fragment is cloned in the productive orientation intothe HindIII site of the plasmid pYG105,which generates the expressionplasmid pYG1206.

Useful variants of the plasmid pET-8c52K are deleted by site-directedmutagenesis between the peptides G10 and G5,for example sites forbinding to collagen, and/or to heparin, and/or to botrocetin, and/or tosulphatides and/or to ristocetin. One example is the plasmid pMMB9deleted by site-directed mutagenesis between the residues Cys509 andIle662. The PCR amplification of this plasmid with theoligodeoxynucleotides Sq1969 and Sq2029 generates an MstII-HindIIIrestriction fragment (FIG. 4, panel D) including the Thr470 to Tyr508and Arg663 to Val713 residues and in particular the peptides G10 and D5of vWF and deleted in particular of its site for binding to collagenlocalized between the residues Glu542 and Met622 [Roth G. J. et al.,Biochemistry 25 (1986) 8357-8361]. The ligation of this fragment to theHindIII-MstII restriction fragment corresponding to the entire geneencoding HSA, with the exception of the three C-terminalmost amino acids(cf. FIG. 2) generates a HindIII restriction fragment containing ahybrid gene encoding a chimeric protein of the HSA-PEPTIDE type (FIG. 1,panel A), immediately preceded by the “prepro” export region of HSA.This restriction fragment is cloned in the productive orientation intothe HindIII site of the plasmid pYG105,which generates the expressionplasmid pYG1223.

In other embodiments, the use of combined techniques of site-directedmutagenesis and PCR amplification makes it possible to generate at willvariants of the MstII-HindIII restriction fragment of panel A of FIG. 4but deleted of one or more sites for binding to sulphatides and/or tobotrocetin and/or to heparin and/or to collagen, and/or substituted byany residue involved in the vWF-associated emergence of IIB typepathologies.

In other useful variants of the plasmid pET-8c52K, mutations areintroduced, for example by site-directed mutagenesis, in order toreplace or suppress all or part of the set of cysteines present atpositions 471, 474, 509 and 695 of the human vWF. Specific examples arethe plasmids p5E and p7E in which the cysteins present at positions 471and 474,on the one hand, and at positions 471, 474, 509 and 695, on theother hand, have been respectively replaced by glycine residues. The PCRamplification of these plasmids with the oligodeoxynucleotides Sq2149(5′-CCCGGGATCCCTTAGGCTTAACCGGTGAAGCCGGC-3′ (SEQ ID NO:28), the MstIIsite is underlined) and Sq2029 makes it possible to generateMstII-HindIII restriction fragments including the Thr470 to Val713residues of the natural vWF with the exception that at least the cysteinresidues at positions 471 and 474 were mutated to glycine residues. Theligation of these fragments to the HindIII-MstII restriction fragmentcorresponding to the entire gene encoding HSA with the exception of thethree C-terminalmost amino acids (cf. FIG. 2) generates a HindIIIrestriction fragment containing a hybrid gene encoding a chimericprotein of the HSA-PEPTIDE type (FIG. 1, panel A), immediately precededby the “prepro” export region of HSA. These restriction fragments arecloned in the productive orientation into the HindIII site of theplasmid pYG105, which generates the expression plasmids pYG1283 (chimeraHSA-vWF470-713, C471G, C474G) and pYG1279 (chimera HSA-vWF470-713,C471G, C474G, C509G, C695G).

Other particularly useful mutations affect at least one residue involvedin vWF-associated type IIB pathologies (increase in the intrinsicaffinity of vWF for GP1b), such as the residues Arg543, Arg545, Trp550,Val551, Val553, Pro574 or Arg578 for example. The genetic recombinationtechniques in vitro also make it possible to introduce at will one ormore additional residues into the sequence of vWF and for example asupernumerary methionine between positions Asp539 and Glu542.

E.7.2. Fragments Antagonizing the Binding of vWF to the Sub-Endothelium

In a specific embodiment, the sites for binding of vWF to the componentsof the sub-endothelial tissue, and for example collagen, are generatedby PCR amplification of the plasmid pET-8c52K, for example with theoligodeoxynucleotides Sq2258(5′-GGATCCTTAGGGCTGTGCAGCAGGCTACTGGACCTGGTC-3′ (SEQ ID NO:29), the MstIIsite is underlined) and Sq2259(5′-GAATTCAAGCTTAACAGAGGTAGCTAA-CGATCTCGTCCC-3′ (SEQ ID NO:30), theHindIII site is underlined), which generates an MstII-HindIIIrestriction fragment encoding the Cys509 to Cys695 residues of thenatural vWF. Deletion molecular variants or modified variants are alsogenerated which contain any desired combination between the sites forbinding of vWF to the sulphatides and/or to botrocetin and/or to heparinand/or to collagen and/or any residue responsible for a modification ofthe affinity of vWF for GP1b (vWF-associated type II pathologies). Inanother embodiment, the domain capable of binding to collagen may alsocome from the vWF fragment which is between the residues 911 and 1114and described by Pareti et al. [J. Biol. Chem. (1987) 262: 13835-13841].The ligation of these fragments to the HindIII-MstII restrictionfragment corresponding to the entire gene encoding HSA with theexception of the three C-terminalmost amino acids (cf. FIG. 2) generatesHindIII restriction fragments containing a hybrid gene encoding achimeric protein of the HSA-PEPTIDE type (FIG. 1, panel A), immediatelypreceded by the “prepro” export region of HSA. These restrictionfragments are cloned in the productive orientation into the HindIII siteof the plasmid pYG105, which generates the corresponding expressionplasmids, and for example the plasmid pYG1277 (HSA-vWF509-695).

E.7.3. Purification and Molecular Characterization of the ChimerasBetween HSA and vWF

The chimeras present in the culture supernatants corresponding to theCBS 293.91 strain transformed, for example with the expression plasmidsaccording to Examples E.7.1. and E.7.2., are characterized in a firstinstance by means of antibodies specific for the HSA part and for thevWF part. The results of FIGS. 5 to 7 demonstrate that the yeast K.lactis is capable of secreting chimeric proteins between HSA and afragment of vWF, and that these chimeras are immunologically reactive.It may also be desirable to purify some of these chimeras. The cultureis then centrifuged (10,000 g, 30 min), the supernatant is passedthrough a 0.22 mm filter (Millipore) and then concentrated byultrafiltration (Amicon) using a membrane whose discrimination thresholdis situated at 30 kDa. The concentrate obtained is then dialysed againsta Tris-HCl solution (50 mM pH 8) and then purified on a column. Forexample, the concentrate corresponding to the culture supernatant of theCBS 293.91 strain transformed with the plasmid pYG1206 is purified byaffinity chromatography on Blue-Trisacryl (IBF). A purification byion-exchange chromatography can also be used. For example, in the caseof the chimera HSA-vWF470-713, the concentrate obtained afterultrafiltration is dialysed against a Tris-HCl solution (50 mM pH 8),and then loaded in 20 ml fractions onto a cation-exchange column (5 ml)(S Fast Flow, Pharmacia) equilibrated in the same buffer. The column isthen washed several times with the Tris-HCl solution (50 mM pH 8) andthe chimeric protein is then eluted from the column by an NaCl gradient(0 to 1M). The fractions containing the chimeric protein are then pooledand dialysed against a 50 mM Tris-HCl solution (pH 8) and then reloadedonto the S Fast Flow column. After elution of the column, the fractionscontaining the protein are pooled, dialysed against water andfreeze-dried before characterization: for example, sequencing (AppliedBiosystem) of the protein [HSA-vWF470-704 C471G, C474G] secreted by theyeast CBS 293.91 gives the N-terminal sequence expected for HSA(Asp-Ala-His . . . ), demonstrating a correct maturation of the chimeraimmediately at the C-terminus of the doublet of residues Arg-Arg of the“pro” region of HSA (FIG. 2). The essentially monomeric character of thechimeric proteins between HSA and vWF is also confirmed by their elutionprofile on a TSK 3000 column [Toyo Soda Company, equilibrated with acacodylate solution (pH 7) containing 0.2M Na2 SO4]: for example thechimera [HSA-vWF 470-704 C471G, C474G] behaves under the conditions likea protein with an apparent molecular weight of 95 kDa, demonstrating itsmonomeric character.

Example 8 Chimeras Derived from Urokinase

E.8.1. Constructs

A fragment corresponding to the amino-terminal fragment of urokinase(ATF: EGF-like domain+ringle domain) can be obtained from thecorresponding messenger RNA of cells of certain human carcinoma, forexample using the RT-PCR kit distributed by Pharmacia. An MstII-HindIIIrestriction fragment including the ATF of human urokinase is given inFIG. 8. The ligation of the HindIII-MstII fragment of the plasmid pYG404to this MstII-HindIII fragment makes it possible to generate the HindIIIfragment of the plasmid pYG1341 which encodes a chimeric protein inwhich the HSA molecule is genetically coupled to the ATF (HSA-UK1→135).Likewise, the plasmid pYG1340 contains a HindIII fragment encoding achimera composed of HSA immediately followed by the first 46 residues ofhuman urokinase (HSA-UK1→46, cf. FIG. 8). The cloning in the productiveorientation, of the HindIII restriction fragment of the plasmid pYG1340(HSA-UK1→46) into the HindIII site of the plasmids pYG105 (LAC4) andpYG106 (PGK) generates the expression plasmids pYG1343 and pYG1342respectively. Likewise, the cloning, in the productive orientation, ofthe HindIII restriction fragment of the plasmid pYG1341 (HSA-UK1→135)into the HindIII site of the plasmids pYG105 (LAC4) and pYG106 (PGK)generates the expression plasmids pYG1345 and pYG1344 respectively.

E.8.2. Secretion of the Hybrids

After selection on rich medium supplemented with G418, the recombinantclones are tested for their capacity to secrete the mature form of thechimeric proteins HSA-UK. A few clones corresponding to the strain K.lactis CBS 293.91, which is transformed with the expression plasmidsaccording to Example E.9.1., are incubated in selective complete liquidmedium at 28° C. The cellular supernatants are then tested afterelectrophoresis on an 8.5% acrylamide gel, either directly by stainingof the gel with coomassie blue, or after immunoblotting using as primaryantibodies a rabbit polyclonal serum directed against human albumin oragainst human urokinase. The results of FIG. 9 demonstrate that thehybrid proteins HSA-UK1→46 and HSA-UK1→135 are particularly wellsecreted by the yeast Kluyveromyces.

E.8.3 Purification of the Chimeras between HSA and Urokinase

After centrifugation of a culture of the CBS 293.91 strain transformedwith the expression plasmids according to Example E.8.1., the culturesupernatant is passed through a 0.22 mm filter (Millipore) and thenconcentrated by ultrafiltration (Amicon) using a membrane whosediscrimination threshold is situated at 30 kDa. The concentrate obtainedis then adjusted to 50 mM Tris-HCl starting with a stock solution of 1MTris-HCl (pH 7), and then loaded in 20 ml fractions onto ananion-exchange column (3 ml) (D-Zephyr, Sepracor) equilibrated in thesame buffer. The chimeric protein (HSA-UK1→46 or HSA-UK1→135) is theneluted from the column by a gradient (0 to 1M) of NaCl. The fractionscontaining the chimeric protein are then pooled and dialysed against a50 mM Tris-HCl solution (pH 6) and reloaded onto a D-Zephyr columnequilibrated in the same buffer. After elution of the column, thefractions containing the protein are pooled, dialysed against water andfreeze-dried before characterization of their biological activity andespecially with respect to their ability to displace urokinase from itscellular receptor.

Example 9 Chimeras Derived from G-CSF

E.9.1. Constructs

E.9.1.1. Coupling at the C-terminus of HSA.

An MstII-HindIII restriction fragment including the mature form of humanG-CSF is generated, for example according to the following strategy: aKpnI-HindIII restriction fragment is first obtained by the enzymatic PCRamplification technique using the oligodeoxynucleotides Sq2291(5′-CAAGGATCC-AAGCTTCAGGGCTGCGCAAGGTGGCGTAG-3′ (SEQ ID NO:31), theHindIII site is underlined) and Sq2292(5′-CGGGGTACCTTAGGCTTAACCCCCCTG-GGCCCTGCCAGC-3′ (SEQ ID NO:32), the KpnIsite is underlined) as primer on the plasmid BBG13 serving as template.The plasmid BBG13 contains the gene encoding the B form (174 aminoacids) of mature human G-CSF, which is obtained from BritishBio-technology Limited, Oxford, England. The enzymatic amplificationproduct of about 550 nucleotides is then digested with the restrictionenzymes KpnI and HindIII and cloned into the vector pUC19 cut with thesame enzymes, which generates the recombinant plasmid pYG1255. Thisplasmid is the source of an MstII-HindIII restriction fragment whichmakes it possible to fuse G-CSF immediately downstream of HSA (chimeraHSA-G.CSF) and whose nucleotide sequence is given in FIG. 10.

It may also be desirable to insert a peptide linker between the HSA partand G-CSF, for example in order to permit a better functionalpresentation of the transducing part. An MstII-HindIII restrictionfragment is for example generated by substitution of the MstII-ApaIfragment of the plasmid pYG1255 by the oligodeoxynucleotides Sq2742(5′-TTAGGCTTAGGTGGTGGCGGT-ACCCCCCTGGGCC-3′ (SEQ ID NO:33), the codonsencoding the glycine residues of this particular linker are underlined)and Sq2741 (5′-CAGGGGGGTACCGCCACCACCTAAGCC-3′) (SEQ ID NO:34) whichform, by pairing, an MsII-ApaI fragment. The plasmid thus generatedtherefore contains an MstII-HindIII restriction fragment whose sequenceis identical to that of FIG. 10 with the exception of the MstII-ApaIfragment.

The ligation of the HindIII-MstII fragment of the plasmid pYG404 to theMstII-HindIII fragment of the plasmid pYG1255 makes it possible togenerate the HindIII fragment of the plasmid pYG1259 which encodes achimeric protein in which the B form of the mature G-CSF is positionedby genetic coupling in translational phase at the C-terminus of the HSAmolecule (HSA-G.CSF).

An identical HindIII restriction fragment, with the exception of theMstII-ApaI fragment, may also be easily generated and which encodes achimeric protein in which the B form of the mature G-CSF is positionedby genetic coupling in translational phase at the C-terminus of the HSAmolecule and a specific peptide linker. For example, this linkerconsists of 4 glycine residues in the HindIII fragment of the plasmidpYG1336 (chimera HSA-Gly4-G.CSF).

The HindIII restriction fragment of the plasmid pYG1259 is cloned in theproductive orientation and into the HindIII restriction site of theexpression plasmid pYG105, which generates the expression plasmidpYG1266 (HSA-G.CSF). In another exemplification, the cloning of theHindIII restriction fragment of the plasmid pYG1259 in the productiveorientation and into the HindIII site of the plasmid pYG106 generatesthe plasmid pYG1267. The plasmids pYG1266 and pYG1267 are mutuallyisogenic with the exception of the SalI-HindIII restriction fragmentencoding the LAC4 promoter of K. lactis (plasmid pYG1266) or the PGKpromoter of S. cerevisiae (plasmid pYG1267).

In another exemplification, the cloning in the productive orientation ofthe HindIII restriction fragment of the plasmid pYG1336 (chimeraHSA-Gly4-G.CSF) into the HindIII site of the plasmids pYG105 (LAC4) andpYG106 (PGK) generates the expression plasmids pYG1351 and pYG1352respectively.

E.9.1.2. Coupling at the N-terminus of HSA.

In a specific embodiment, the combined techniques of site-directedmutagenesis and PCR amplification make it possible to construct hybridgenes encoding a chimeric protein resulting from the translationalcoupling between a signal peptide (and for example the prepro region ofHSA), a sequence including a gene having a G-CSF activity, and themature form of HSA or one of its molecular variants (cf. chimera ofpanel B, FIG. 1). These hybrid genes are preferably bordered in 5′ ofthe translational initiator ATG and in 3′ of the translational stopcodon by HindIII restriction sites. For example the oligodeoxynucleotideSq2369 (5′-GTTCTACGCCACCTTGCGCAGCCCGGTGGAGGCGGTGATGCACACAAGAGTGAGGTTGCTCATCGG-3′ (SEQ ID NO:35) the residues underlined (optional)correspond in this particular chimera to a peptide linker composed of 4glycine residues) makes it possible, by site-directed mutagenesis, toput in translational phase the mature form of the human G-CSF of theplasmid BBG13 immediately upstream of the mature form of HSA, whichgenerates the intermediate plasmid A. Likewise, the use of theoligodeoxynucleotide Sq2338[5′-CAGGGAGCTGGCAGGGCCCAGGGGGGTTCGACGAAACACACCCCTGGAATAAGCC GAGCT-3′(SEQ ID NO:36) (non-coding strand), the nucleotides complementary to thenucleotides encoding the first N-terminal residues of the mature form ofthe human G-CSF are underlined] makes it possible, by site-directedmutagenesis, to couple in translational reading phase the prepro regionof HSA immediately upstream of the mature form of the human G-CSF, whichgenerates the intermediate plasmid B. A HindIII fragment encoding achimeric protein of the PEPTIDE-HSA type (cf. FIG. 1, panel B) is thengenerated by combining the HindIII-SstI fragment of the plasmid B(joning prepro region of HSA+N-terminal fragment of the mature G-CSF)with the SstI-HindIII fragment of the plasmid A [joining matureG-CSF-(glycine)×4-mature HSA]. The plasmid pYG1301 contains thisspecific HindIII restriction fragment encoding the chimeraG.CSF-Gly4-HSA fused immediately downstream of the prepro region of HSA(FIG. 11). The cloning of this HindIII restriction fragment in theproductive orientation and into the HindIII site of the plasmids pYG105(LAC4) and pYG106 (PGK) generates the expression plasmids pYG1302 andpYG1303 respectively.

E.9.2. Secretion of the Hybrids

After selection on rich medium supplemented with G418, the recombinantclones are tested for their capacity to secrete the mature form of thechimeric proteins between HSA and G-CSF. A few clones corresponding tothe strain K. lactis CBS 293.91 transformed with the plasmids pYG1266 orpYG1267 (HSA-G.CSF), pYG1302 or pYG1303 (G.CSF-Gly4-HSA) oralternatively pYG1351 or pYG1352 (HSA-Gly4-G.CSF) are incubated inselective complete liquid medium at 28° C. The cellular supernatants arethen tested after electrophoresis on an 8.5% acrylamide gel, eitherdirectly by staining the gel with coomassie blue, or afterimmunoblotting using as primary antibodies rabbit polyclonal antibodiesdirected against the human G-CSF or a rabbit polyclonal serum directedagainst human albumin. The results of FIG. 12 demonstrate that thehybrid protein HSA-G.CSF is recognized both by antibodies directedagainst human albumin (panel C) and human G-CSF (panel B). The resultsof FIG. 13 indicate that the chimera HSA-Gly4-G.CSF (lane 3) isparticularly well secreted by the yeast Kluyveromyces, possibly becauseof the fact that the presence of the peptide linker between the HSA partand the G-CSF part is more favourable to an independent folding of these2 parts during the transit of the chimera in the secretory pathway.Furthermore, the N-terminal fusion (G.CSF-Gly4-HSA) is also secreted bythe yeast Kluyveromyces (FIG. 13, lane 1).

E.9.3. Purification and Molecular Characterization of the Chimerasbetween HSA and G-CSF

After centrifugation of a culture of the CBS 293.91 strain transformedwith the expression plasmids according to Example E.9.1., the culturesupernatant is passed through a 0.22 mm filter (Millipore) and thenconcentrated by ultrafiltration (Amicon) using a membrane whosediscrimination threshold is situated at 30 kDa. The concentrate obtainedis then adjusted to 50 mM Tris-HCl from a 1M stock solution of Tris-HCl(pH 6), and then loaded in 20 ml fractions onto an ion-exchange column(5 ml) (Q Fast Flow, Pharmacia) equilibrated in the same buffer. Thechimeric protein is then eluted from the column by a gradient (0 to 1M)of NaCl. The fractions containing the chimeric protein are then pooledand dialysed against a 50 mM Tris-HCl solution (pH 6) and reloaded ontoa Q Fast Flow column (1 ml) equilibrated in the same buffer. Afterelution of the column, the fractions containing the protein are pooled,dialysed against water and freeze-dried before characterization: forexample, the sequencing (Applied Biosystem) of the protein HSA-G.CSFsecreted by the yeast CBS 293.91 gives the N-terminal sequence expectedfor HSA (Asp-Ala-His . . . ), demonstrating a correct maturation of thechimera immediately at the C-terminus of the doublet of residues Arg-Argof the “pro” region of HSA (FIG. 2).

Example 10 Chimeras Derived from an Immunoglobulin

E.10.1. Constructs

An Fv′ fragment can be constructed by genetic engineering techniques,and which encodes the variable fragments of the heavy and light chainsof an immunoglobulin (Ig), linked to each other by a linker peptide[Bird et al., Science (1988) 242: 423; Huston et al., (1988) Proc. Natl.Acad. Sci. 85: 5879]. Schematically, the variable regions (about 120residues) of the heavy and light chains of a given Ig are cloned fromthe messenger RNA of the corresponding hybridoma, for example using theRT-PCR kit distributed by Pharmacia (Mouse ScFv module). In a secondstage, the variable regions are genetically coupled by geneticengineering via a synthetic linkage peptide and for example the linker(GGGGS)×3. An MstII-HindIII restriction fragment including the Fv′fragment of an immunoglobulin secreted by a murine hybridoma is given inFIG. 14. The ligation of the HindIII-MstII fragment of the plasmidpYG404 to this MstII-HindIII fragment makes it possible to generate theHindIII fragment of the plasmid pYG1382 which encodes a chimeric proteinin which the HSA molecule is genetically coupled to the Fv′ fragment ofFIG. 14 (chimera HSA-Fv′). The cloning in the productive orientation ofthe HindIII restriction fragment of the plasmid pYG1382 into the HindIIIsite of the plasmids pYG105 (LAC4) and pYG106 (PGK) generates theexpression plasmids pYG1383 and pYG1384 respectively.

E. 10.2. Secretion of the Hybrids

After selection on rich medium supplemented with G418, the recombinantclones are tested for their capacity to secrete the mature form of thechimeric protein HSA-Fv′. A few clones corresponding to the strain K.lactis CBS 293.91 transformed with the plasmids pYG1383 or pYG1384(HSA-Fv′) are incubated in selective complete liquid medium at 28° C.The cellular supernatants are then tested after electrophoresis on an8.5% acrylamide gel, either directly by staining of the gel withcoomassie blue, or after immunoblotting using as primary antibodies arabbit polyclonal serum directed against human albumin, or directlyincubated with biotinylated antibodies directed against theimmunoglobulins of murine origin. The results of FIG. 15 demonstratethat the hybrid protein HSA-Fv′ is recognized both by antibodiesdirected against human albumin (panel C) and reacts with biotinylatedgoat antibodies which are immunologically reactive towards mouseimmunoglobulins (panel B).

Example 11 Biological Activity of the Chimeras

E.11.1. Biological Activity In Vitro

E.11.1.1. Chimeras between HSA and vWF

The antagonistic activity of the products is determined by measuring thedose-dependent inhibition of the agglutination of human platelets fixedwith paraformaldehyde according to the method described by Prior et al.[Bio/Technology (1992) 10: 66]. The measurements are carried out in anaggregameter (PAP-4, Bio Data, Horsham, Pa., U.S.A.) which records thevariations over time of the optical transmission, with stirring, at 37°C. in the presence of vWF, of botrocetin (8.2 mg/ml) and of the testproduct at various dilutions (concentrations). For each measurement, 400ml (8×10⁷ platelets) of a suspension of human platelets stabilized withparaformaldehyde (0.5%, and then resuspended in [NaCl (137 mM); MgCl2 (1mM); NaH2 PO4 (0.36 mM); NaHCO3 (10 mM); KCl (2.7 mM); glucose (5.6 mM);HSA (3.5 mg/ml); HEPES buffer (10 mM, pH 7.35)] are preincubated at 37°C. in the cylindrical tank (8.75×50 mm, Wellcome Distriwell, 159 rueNationale, Paris) of the aggregameter for 4 min and are thensupplemented with 30 ml of the solution of the test product at variousdilutions in apyrogenic formulation vehicle [mannitol (50 g/l); citricacid (192 mg/l); L-lysine monohydrochloride (182.6 mg/l); NaCl (88mg/l); pH adjusted to 3.5 by addition of NaOH (1M)], or formulationvehicle alone (control assay). The resulting suspension is thenincubated for 1 min at 37° C. and 12.5 ml of human vWF [AmericanBioproducts, Parsippany, N.J., U.S.A.; 11% von Willebrand activitymeasured according to the recommendations for the use of PAP-4 (PlateletAggregation Profiler®) with the aid of platelets fixed with formaldehyde(2×10⁵ platelets/ml), human plasma containing 0 to 100% vWF andristocetin (10 mg/ml, cf. p. 36-45: vW Program™] are added and incubatedat 37° C. for 1 min before adding 12.5 ml of botrocetin solution[purified from freeze-dried venom of Bothrops jararaca (Sigma) accordingto the procedure described by Sugimoto et al., Biochemistry (1991) 266:18172]. The recording of the reading of the transmission as a functionof time is then carried out for 2 min with stirring by means of amagnetic bar (Wellcome Distriwell) placed in the tank and with amagnetic stirring of 1,100 rpm provided by the aggregameter. The meanvariation of the optical transmission (n3 5 for each dilution) over timeis therefore a measurement of the platelet agglutination due to thepresence of vWF and botrocetin, in the absence or in the presence ofvariable concentrations of the test product. From such recordings, the %inhibition of the platelet agglutination due to each concentration ofproduct is then determined and the straight line giving the % inhibitionas a function of the reciprocal of the product dilution in log-log scaleis plotted. The IC50 (or concentration of product causing 50% inhibitionof the agglutination) is then determined on this straight line. Thetable of FIG. 6 compares the IC50 values of some of the HSA-vWF chimerasof the present invention and demonstrates that some of them are betterantagonists of platelet agglutination than the product RG12986 describedby Prior et al. [Bio/Technology (1992) 10: 66] and included in theassays as standard value. Identical tests for the inhibition of theagglutination of human platelets in the presence of vWF of pig plasma(Sigma) makes it possible, furthermore, to demonstrate that some of thehybrids of the present invention, and especially some type IIB variants,are very good antagonists of platelet agglutination in the absence ofbotrocetin-type cofactors. The botrocetin-independent antagonism ofthese specific chimeras can also be demonstrated according to theprocedure initially described by Ware et al. [Proc. Natl. Acad. Sci.(1991) 88: 2946] by displacing the monoclonal antibody 125 I-LJ-IB1 (10mg/ml), a competitive inhibitor of the binding of vWF to the plateletGP1b [Handa M. et al., (1986) J. Biol. Chem. 261: 12579] after 30 min ofincubation at 22° C. in the presence of fresh platelets (108platelets/ml).

E.11.1.2. Chimeras between HSA and G-CSF

The purified chimeras are tested for their capacity to permit the invitro proliferation of the IL3-dependant murine line NFS60,by measuringthe incorporation of tritiated thymidine essentially according to theprocedure described by Tsuchiya et al. [Proc. Natl. Acad. Sci. (1986) 837633]. For each chimera, the measurements are carried out between 3 and6 times in a three-point test (three dilutions of the product) in a zoneor the relation between the quantity of active product and incorporationof labelled thymidine (Amersham) is linear. In each microtitre plate,the activity of a reference product consisting of recombinant humanG-CSF expressed in mammalian cells is also systematically incorporated.The results of FIG. 17 demonstrate that the chimera HSA-G.CSF (pYG1266)secreted by the yeast Kluyveromyces and purified according to ExampleE.9.3. is capable in vitro of transducing a signal for cellularproliferation for the line NFS60. In this particular case, the specificactivity (cpm/molarity) of the chimera is about 7 times lower than thatof the reference G-CSF (non-coupled).

E.11.2. Biological Activity In Vivo

The activity of stimulation of the HSA-G-CSF chimeras on granulopoiesisin vivo is tested after subcutaneous injection in rats(Sprague-Dawley/CD, 250-300 g, 8-9 weeks) and compared to that of thereference G-CSF expressed using mammalian cells. Each product, tested atthe rate of 7 animals, is injected subcutaneously into thedorso-scapular region at the rate of 100 ml for 7 consecutive days,(D1-D7). 500 ml of blood are collected on days D-6, D2 (before the 2ndinjection). D5 (before the 5th injection) and D8, and a blood count isperformed. In this test, the specific activity (neutropoiesis units/moleinjected) of the chimera HSA-G.CSF (pYG1266) is identical to that of thereference G-CSF (FIG. 18). Since this specific chimera has in vitro aspecific activity 7 times lower than that of the reference G-CSF (FIG.17), it is therefore demonstrated that the genetic coupling of G-CSFonto HSA favourably modifies the pharmacokinetic properties thereof.

1. method of treating a patient in need of erythropoietin, comprisingthe step of administering a fusion protein comprising erythropoietin andalbumin or an albumin variant, wherein (i) said fusion protein has ahigher plasma stability than unfused erythropoietin, (ii) said fusionprotein retains the therapeutic activity of unfused erythropoietin, and(iii) said albumin or albumin variant is located either at theN-terminus or C-terminus of said fusion protein.
 2. The method of claim1, wherein said fusion protein comprises albumin.
 3. The method of claim1, wherein said fusion protein comprises an albumin variant.
 4. Themethod of claim 3, wherein said albumin variant is a fragment ofalbumin.
 5. The method of claim 3, wherein said albumin variant has amutation of one or more residues.
 6. The method of claim 3, wherein saidalbumin variant has a deletion of one or more residues.
 7. The method ofclaim 3, wherein said albumin variant has a mutation and a deletion ofone or more residues.
 8. The method of claim 3, wherein said albuminvariant has an addition of one or more residues.
 9. The method of claim1, wherein said fusion protein comprises an N-terminal methionine. 10.The method of claim 1, wherein said fusion protein comprises a peptidelinker.
 11. The method of claim 1, wherein said fusion protein comprisesa secretion signal sequence.
 12. The method of claim 11, wherein saidsecretion signal sequence is the natural leader sequence oferythropoietin.
 13. The method of claim 1, wherein said erythropoietinis fused to the N-terminal end of said albumin or albumin variant. 14.The method of claim 1, wherein said erythropoietin is fused to theC-terminal end of said albumin or albumin variant.
 15. A method oftreating a patient in need of erthyropoietin, comprising the step ofadministering a fusion protein comprising erythropoietin and a matureform of albumin, wherein (i) said fusion protein has a higher plasmastability than unfused erythropoietin, (ii) said fusion protein retainsthe therapeutic activity of unfused erythropoietin, and (iii) saidalbumin or albumin variant is located either at the N-terminus orC-terminus of said fusion protein.
 16. The method of claim 15, whereinsaid fusion protein comprises an N-terminal methionine.
 17. The methodof claim 15, wherein said fusion protein comprises a peptide linker. 18.The method of claim 15, wherein said fusion protein comprises asecretion signal sequence.
 19. The method of claim 18, wherein saidsecretion signal sequence is the natural leader sequence oferythropoietin.
 20. The method of claim 15, wherein said erythropoietinis fused to the N-terminal end of said mature form of albumin.
 21. Themethod of claim 20, wherein said fusion protein comprises an N-terminalmethionine.
 22. The method of claim 20, wherein said fusion proteincomprises a peptide linker.
 23. The method of claim 20, wherein saidfusion protein comprises a secretion signal sequence.
 24. The method ofclaim 23, wherein said secretion signal sequence is the natural leadersequence of erythropoietin.
 25. The method of claim 15, wherein saiderythropoietin is fused to the C-terminal end of said mature form ofalbumin.
 26. The method of claim 25, wherein said fusion proteincomprises an N-terminal methionine.
 27. The method of claim 25, whereinsaid fusion protein comprises a peptide linker.
 28. The method of claim25, wherein said fusion protein comprises a secretion signal sequence.29. A The method of claim 28, wherein said secretion signal sequence isthe natural leader sequence of erythropoietin.